1 R21 AI A1 2 VMD HALFORD, W

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2 1 R21 AI A1 2 VMD 1R21AI A1 ILLIAM RESUME AND SUMMARY OF DISCUSSION: The proposed study is to develop safe and effective live attenuated vaccines against herpes simplex virus 2 by using several animal models. The objective is highly significant; if successful, the proposed work could impact on the field of herpes vaccine development. The investigator is responsive to the concerns raised by the previous reviewers and the application is improved. During discussion, reviewers noted many strengths which include the new preliminary data are supportive, the approach is innovative, the plan to use mouse and guinea pig models is sound, and the investigator is well qualified for the proposed study. However, reviewers also identified several concerns, some of which were raised in prior reviews. There is no assessment of the durability of the proposed vaccine. The numbers of animals will be used might not be sufficient for statistical analysis. Also, the rationale for using rabbits and hamsters is not very clear and the use of multiple animal species is beyond the scope of an R21 grant mechanism. In addition, the disease monitoring system is not included. Overall, the review committee expressed moderate enthusiasm for this otherwise interesting application. DESCRIPTION (provided by applicant): For 30 years, an effective vaccine has been sought to prevent genital herpes caused by herpes simplex virus 2 (HSV-2). Most of the research has focused on the development of non-replicating HSV-2 vaccine agents such as the glycoprotein D subunit or replication-defective HSV-2 viruses. Such approaches merit consideration. However, it is unclear that non-replicating HSV-2 vaccines elicit the type of life-long immunity against genital herpes that is sought. For each year that elapses without an effective vaccine, another 10 to 20 million people contract HSV-2 infections. Given the scope of the problem, perhaps it is time to consider a second possibility: a live, replicating HSV-2 vaccine strain may be more effective. Most of our successes in controlling viral disease in the human population have been based upon live, replicating viruses. Live vaccinia virus (the original vaccine) was used to end smallpox epidemics. Poliomyelitis, mumps, measles, rubella, and chickenpox are prevented with childhood vaccines that contain live, replicating viruses that occur in nature, but are attenuated in their disease-causing potential. This, our most successful vaccination modality, has not been adequately considered for its potential to control genital herpes. In large part, this is due to the misconception that a live HSV-2 vaccine strain would be dangerous. The P.I.'s published studies and preliminary data establish that genetic engineering combined with current knowledge of HSV biology may be applied to derive live, replicating HSV-1 and HSV-2 viral vaccines that are safe and immunogenic. In principle, attenuation of HSV is readily achieved because ~30 of 75 HSV genes are not essential for viral replication. Many of these genes, such as the ICP0 gene, are required for HSV to resist repression by the host immune response. The P.I. has worked with interferon-sensitive HSV-1 ICP0- viruses for 10 years. Disruption of the ICP0 gene renders HSV-1 and HSV-2 hypersensitive to repression by interferon-1/2, avirulent in animals, and yet these viruses may serve as powerful immunogens. Mice immunized with a live, replicating HSV-1 ICP0- virus are immune to lethal challenge with 1000 times the LD50 dose of HSV-1 (McKrae strain) or HSV-2 (MS strain). Likewise, mice in which an HSV-2 ICP0- virus replicates at the site of immunization are immune to lethal challenge with HSV-2 MS. A systematic effort has not been made to develop a live and appropriately attenuated HSV-2 virus that may establish an inapparent infection at the site of immunization. The P.I.'s data indicate that HSV-1 or HSV-2's full immunogenic potential is only realized when viral replication occurs in the host. If this hypothesis is correct, then live, replicating HSV vaccine strains may be far more protective than any non-replicating HSV vaccine considered to date. Two years of R21 funding is requested to test this hypothesis, and to begin developing HSV-2 ICP0- viruses that may later be used in human clinical trials if a live HSV-2 vaccine strain proves to be safe and effective in protecting mice, guinea pigs, rabbits, and hamsters against genital herpes. PUBLIC HEALTH RELEVANCE: Interferon-sensitive herpes simplex virus-2 (HSV-2) ICP0- viruses are proposed as a live, replicating HSV-2 vaccine strain. Such live HSV-2 vaccine strains may be capable of preventing the spread of genital herpes, a disease that currently afflicts ~50 million people worldwide. The work proposed herein will test a hypothesis that live, replicating HSV-2 ICP0- viruses provide superior protection against exogenous HSV-2 infections relative to non-replicating HSV-2 vaccines that have

3 1 R21 AI A1 3 VMD been the focus of research for 30 years. It is anticipated that a new live HSV-2 ICP0- vaccine strain will emerge from these studies that warrants advancement to human clinical trials. CRITIQUE 1: Significance: There has been much work but little success over the last few decades trying to develop a successful herpes simplex virus (HSV) vaccine. Subunit approaches have led to vaccines that were only partially protective. Clearly the development of a successful HSV2 vaccine would be highly significant and of great medical value. This application proposes a unique way to re-explore the possibility of developing live, mutant viruses that could have the appropriate balance between attenuation and immunogenicity to be useful vaccine candidates. While significant safety concerns will continue to be raised toward an approach like this one that uses a virus that establishes latency, successful development of these vaccine candidates could impact the herpes vaccine field. Progress, Changes, and Responses to the Previous Summary Statement: This is a revised, resubmitted application. Most of the key concerns of the previous reviewers have been addressed including: 1) Explanation of why the attenuated virus strains to be developed should be superior to previously developed strains (balance of attenuation and replication/latency); 2) Adjuvant to be used with the control subunit vaccine (addition of MPL to gd vaccine); 3) Use of ocular route for immunization and challenge (inclusion of footpad and vaginal immunization routes, vaginal challenge route in guinea pig model); 4) Lack of determination of latency (demonstration with HSV1 mutants in preliminary data and addition of this analysis for proposed HSV2 mutants in research plan); 5) Immunological studies are of questionable value (added preliminary data demonstrating correlation of IgG titers and protection); 6) There is no reactivation of HSV in mice (literature data cited that there appears to be limited subclinical reactivation in mice, and in humans); 7) Will the viral mutants establish latent or pathogenic infections? (preliminary data provided that demonstrates the HSV1 ICP0 null mutants establish latent infection and that HSV2 ICP0 null mutants are avirulent in vivo); and 8) Positive control animals treated with wild type HSV2 will die and not be available for study (preliminary data provided that surviving mice with latent wt HSV2 can be established and used as controls). Addressing these concerns has significantly improved the application. Approach: The investigator proposes to develop attenuated HSV2 vaccines that could give potent and long lasting immunity by achieving a balance of replication, latency and immunogenicity. To do this, the investigator will focus on ICP0, a protein that counteracts the host type-1 interferon responses. ICP0 null mutants are dramatically attenuated but the investigator s preliminary data with both HSV1 and more recently HSV2 show that they can establish latency and provide some level of protection against wild type challenge. The proposal involves generating a series of HSV ICP0 mutants that have differing levels of ICP0 activity and test these mutants for pathogenicity, latency, and protective immunity. The hypothesis is that they can establish a mutant virus with an intermediate ICP0 activity between wild type and null that will display the appropriate balance between safety and protective immunogencity that would be useful as an HSV2 vaccine. The investigator proposes to first establish the safety and efficacy of the candidate mutant viruses using the ocular model in the mouse. This involves monitoring latency, pathogenicity, immunogencity, and protective immunity. Subsequently they will move the successful candidates to the guinea pig and test both additional vaccination sites (footpad and vagina) and well as testing both ocular and vaginal challenge. Finally they propose to test the leading candidates in additional models including rabbits and hamsters to test that the vaccine safety and immunogencity are not species specific. It appears that the plans for developing the HSV2 mutants and testing them in the mouse model are solid. This plan has been strengthened significantly in the revised application by response to the previous reviewers concerns (see above). The plan for testing the candidates in the guinea pig model is also significantly improved by adding the additional immunization and challenge sites. One concern here is that the numbers of animals per group (5) may not be sufficient for statistical comparisons

4 1 R21 AI A1 4 VMD between vaccine candidates. Another concern is that while the proposal hypothesizes that this type of vaccine could provide long lasting or even lifetime immunity, and that this would be a key advantage of a vaccine that establishes latency, there are no experiments proposed that would look at the duration of immunity. Only one time point is proposed for all challenge experiments (day 70). Finally, it is not clear what value expanding the protective studies to rabbits and hamsters provides. If the vaccines are safe and effective in mice and guinea pigs, positive or negative results in these less-developed HSV models will probably not be key factors in determining if the ICP0 HSV2 mutants are ready for clinical testing or not. Innovation: The idea of using ICP0 mutants with differing ICP0 activity to try to achieve an appropriate balance between attenuation and immunogencity is innovative. Investigators: Dr. Halford is an experienced herpes virologist and is highly qualified to conduct the proposed research program. Environment: The environment at Southern Illinois University School of medicine appears appropriate for the proposed research program. Overall Evaluation: This application uses a unique way to re-explore the possibility of developing live, mutant viruses that could have the appropriate balance between attenuation and immunogenicity to be useful vaccine candidates. While safety concerns will continue to be raised toward an approach that uses a virus that establishes latency, successful development of these vaccine candidates could impact the herpes vaccine field. Most of the key concerns of the previous reviewers in this resubmitted proposal have been adequately addressed. It appears that the plans for developing the HSV2 mutants and testing them in the mouse model are solid. This plan has been strengthened significantly in the revised application by response to the previous reviewers concerns. The plan for testing the candidates in the guinea pig model is also significantly improved by adding the additional immunization and challenge sites. One concern is that the numbers of animals per group (5) may not be sufficient for statistical comparisons between vaccine candidates. Another concern is that the proposal hypothesizes that this type of vaccine could provide long lasting or even lifetime immunity, but there are no experiments proposed that would study the duration of immunity. It is not clear what value expanding the protective studies to rabbits and hamsters provides. Protection of Human Subjects from Research Risks: Not applicable. Vertebrate Animals: Acceptable. Biohazards and Select Biological Agents and Toxins: No concerns. Budget: The budget seems appropriate. Foreign Institution: Not applicable. Data Sharing Plan: Acceptable. Model Organism Sharing Plan: Acceptable. CRITIQUE 2: Significance: HSV is a major clinical problem worldwide. In the US alone, over 100 million individuals are infected with HSV-1, and at least 40 million to 60 million individuals are infected with HSV-2. The current studies are directed at developing new live attenuated vaccines against HSV and are therefore considered to be highly significant.

5 1 R21 AI A1 5 VMD Progress, Changes, and Responses to the Previous Summary Statement: See Approach, below. Approach: This is a resubmission of a 2-year R21 application by Dr. William Halford to develop an effective genital herpes vaccine. The underlying principle that the investigator wishes to test is whether a vaccine that establishes a persistent infection would confer lifelong immunity. Evidence that this approach is feasible comes from the VZV vaccine against chicken pox (the vaccine may establish latency in vaccinees) and the fact that many individuals that are latently infected with HSV are asymptomatic. The prior version of this application was generally well received by the reviewers. However, there were several significant concerns regarding the underlying rationale and some aspects of the experimental approach. The application has been significantly reorganized and many of the specific concerns have been directly addressed. Importantly, some new approaches have been included and some of the experimental designs have been improved. These include the utilization of more appropriate adjuvants and the analysis of vaccine efficacy in other animal species (although see comment below). As such, the revised application is considerably improved. A key improvement in the application is a much better explanation of the underlying rationale for the studies and a better integration of the preliminary data with the proposed experimental approach. The immunology is presented in a straightforward and logical fashion and it seems likely that important new data will be generated. In addition, the use of the guinea pig (a more relevant model than mouse) to test vaccine efficacy is a major strength. Less clear is the expansion of the studies to rabbits and hamsters. While there is a need to show efficacy in additional species, it seemed to be outside the scope of this R21 application. Although the immunological aspects of the application were improved, some concerns remained outstanding. Of particular note, the investigator does not address the durability of the vaccine; an important question that could be readily addressed within the experimental design. This issue was raised in the last review, however it was not considered in the revision. A major strength of the application is that the investigator presents new preliminary data illustrating the efficacy of the approach in generating antibodies that bind to HSV-2 antigens. It is noteworthy that antibodies generated with the attenuated virus were superior to those generated by protein vaccination. In addition, the live vaccine exhibited some protective efficacy against a lethal dose challenge whereas the protein vaccinations did not. Innovation: This grant application proposes to use attenuated HSV viral strains that will establish lifelong infection and, theoretically, lifelong immunity to infection by wild-type HSV. This is a highly innovative approach. Investigators: Dr. Halford is an Associate Professor, Department of Medical Microbiology and Immunology at Southern Illinois University School of Medicine, Springfield, IL and will devote 3 calendar months effort to the project. He is an expert in an expert in HSV virology and is highly qualified to direct this project. Environment: The proposed studies will be performed at Southern Illinois University School of Medicine, Springfield, IL. The scientific environment is outstanding. Overall Evaluation: This is an interesting resubmission of a 2-year R21 application by Dr. Halford to develop a live-attenuated genital herpes vaccine. The investigator has done an excellent job of addressing the reviewers concerns by revising some of the experimental designs and by reorganizing, and to some extent, refocusing the application. The revised application is considerably improved. A major strength of the application is that the underlying concept is innovative and the application addresses a problem of great clinical significance. In addition, the virological expertise of the

6 1 R21 AI A1 6 VMD investigator is a significant major strength. Weaknesses of the application include the failure to assess the durability of this vaccine approach and the use of multiple animal species (which was considered to be outside the scope of the application). Protection of Human Subjects from Research Risks: Not applicable. Vertebrate Animals: No concerns. Biohazards And Select Biological Agents And Toxins: Not included, but no concern. Budget: No concerns. Data Sharing Plan: Included. Model Organism Sharing Plan: Not included, but no concern. CRITIQUE 3: Overall Evaluation: This is a revised R21 application to create attenuated strains of HSV-2 and to evaluate their potential as vaccine candidates in animal models. The PI, Dr. Halford is an experienced HSV virologist, and the environment is supportive. The concept of a live attenuated herpes virus vaccine is not entirely novel and indeed Dr. Halford has published and presents preliminary data for similar approaches for HSV-1. The application of the attenuating mutations to ICP0 is new to HSV-2, however. Since HSV-2 is a cause for significant morbidity world-wide, the successful development of a safe and efficacious vaccine would have significant impact and successful completion of this application could eventually lead in that direction. In this revised R21 application the PI has responded to the previous critique by extensive modification. New preliminary data has been added in which approximately 1/3 of the proposed new ICP0 mutant strains of HSV-2 have been constructed and a pilot study using one of these strains exhibited partial protection from HSV-2 shedding and enhanced survival when compared to an HSV-2 subunit vaccine. Other changes and additions to the application have also improved it. The application does retain murine ocular immunization and challenge as an assay to screen the mutant viruses which will be generated, but also goes on to explore more relevant immunization and challenge routes in additional animal species (guinea pig, hamster, and rabbit) after screening is completed. This seems like a reasonable response. The ultimate goal is creating a live attenuated vaccine for HSV-2. Even in pilot studies, safety is an issue and one aspect which still has not been addressed is the potential for the live attenuated virus to cause disease. Although disease is mentioned several times throughout the application, the only endpoints enumerated are virus shedding and animal survival. The central hypothesis is that local replication and probably even establishment of a latent infection will be required for vaccine efficacy. However, no measures of disease, either transient or chronic, which might be induced by infection with live attenuated vaccine strains or resulting from virulent virus challenge after immunization are included. No disease scoring system from daily observation of infected animals is included and no histologic analysis of local or systemic ganglia infections are included. Thus although the application is significantly improved, the lack of disease monitoring remains a weakness. THE FOLLOWING RESUME SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE ON THE FOLLOWING ISSUES: VERTEBRATE ANIMALS (Resume): Acceptable COMMITTEE BUDGET RECOMMENDATIONS: The budget is recommended as requested.

7 1 R21 AI A1 7 VMD NOTICE: In 2008 NIH modified its policy regarding the receipt of resubmission (formerly termed amended) applications. Detailed information can be found by accessing the following URL address: NIH announced implementation of Modular Research Grants in the December 18, 1998 issue of the NIH Guide to Grants and Contracts. The main feature of this concept is that grant applications (R01, R03, R21, R15) will request direct costs in $25,000 modules, without budget detail for individual categories. Further information can be obtained from the Modular Grants Web site at